Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack

Background Aedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection. Methods Colonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively. Results Replicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3–5.2) and An. stephensi (OR 2.7, 95% CI 1.4–5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26–0.62; An. stephensi OR = 0.59, 95% CI 0.36–0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85–1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44–1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species. Conclusions Aedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06364-9.


Introduction
There are 4 main types of effects we are measuring (engorgement rates, hatch rates, oocyst counts per infected midgut, and sporozoites per infected mosquito).We list the data by generation and colony in the main tables of the paper (Tables 1-4).In those Tables we use averages and interquartile ranges throughout.
For hatch rates and oocyst counts, there are two levels of summarization.For example, for hatch rates we first take the average and interquartile range (IQR) of groups of eggs.These are presented for each generation/colony row.Then we take the unweighted average and IQR of those averages within each generation.Oocyst counts averages and IQRs are done similarly.We use IQR throughout Tables 1-4 for consistency, even though it is non-standard to calculate an IQR with only 2-4 observations.For comparing the effects of the meal, bovine blood versus SkitoSnack, the analyses for each of the 4 types of effects are different and necessarily more sophisticated, so that each can properly account for any of the types of variability in its measurement (e.g., variation between colonies, generations, mosquitoes, eggs, oocysts, and sporozoites).We provide the details of those analyses in separate sections of this supplement.These analyses were performed in R version 4.3.0 (2023-04-21).

Analysis Details
• In the sections below we present analyses to determine whether the type of meal (BB: bovine blood or SS: SkitoSnack) has an effect on engorgement rates for Aedes aegypti and Anopheles stephensi.• We consider a logistic regression mixed effects model.
-Random effects: In our work, considered to be factors (or categorical variables) with levels that are a random sample from some population.These are the colony replicates for each generation.-Fixed effects: In our work, considered to be factors with treatments that are pre-specified by the researcher and are constant across individuals (mosquitoes).These are meal and generation.• The models were fit using the package lme4, citation: Bates D, Mächler M, Bolker B, Walker S (2015).
"Fitting Linear Mixed-Effects Models Using lme4."Journal of Statistical Software, 67(1), 1-48.• The 95% confidence intervals for the estimated probabilities were obtained via bootstrap with 1000 iterations.• The reference meal is BB and reference generation is F0.
• A likelihood ratio test (LRT) was performed to assess an interaction between generation and meal.

Aedes aegypti
Engorgement Rate (%) Figure 1: Aedes aegypti engorgement rate (%).Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.
• We performed an LRT to determine whether there was a significant interaction between generation and meal.The p-value of the test is p: 0.895.
• We found a significant effect due to meal.The odds of a mosquito engorging when the meal is SS are 2.6 (95%CI:[1.33,5.23])times higher than when the meal is BB.
• We found a significant effect due to generation from a LRT p: 0.034.This is an overall effect, though each individual test of each generation to F0 may not be significant as displayed on the table below.

Anopheles stephensi
• We performed an LRT to determine whether there was a significant interaction between generation and meal.The p-value of the test is p: 0.023.• Because of our significant interaction results, we performed individual contrasts to test whether there is a significant difference between SS and BB at each generation.• Although the generation effect appears to be significant as shown in the LRT and the above figures, we do not see a meaningful pattern.Therefore, to get an overall averaging estimate of the meal effect we treat the interaction as part of the error.

Analysis Details
• In the sections below we present analyses to determine whether the type of meal (BB: bovine blood or SS: SkitoSnack) has an effect on hatch rate.• We consider a logistic regression with quasibinomial family for our variable of interest.
• We include a generation by meal interaction to estimate the effect of diet at each generation and use a test of deviance to assess whether the interaction is significant.• If the interaction is not significant it is removed from the model.• Reference values: The reference for Meal is BB and for Generation is F0.

Aedes aegypti
Hatch Rate (%) • We did not find sufficient evidence of a significant interaction, test of deviance p: 0.239.
• We found evidence of a significant generation effect, test of deviance p: 0.023.
• We found evidence of significant effect of meal p:<0.001.The odds of an egg hatching are 0.4 times lower (95%CI:[0.26,0.62])when the meal is SkitoSnack when compared to bovine blood.• We found no significant interaction.Test of deviance p: 0.581.
• We found evidence of a significant generation effect.Test of deviance p: <0.001.
• We found evidence of a significant effect of meal p:0.041.The odds of an egg hatching are 0.59 times lower (95%CI:[0.36,0.96])when the meal is SkitoSnack when compared to bovine blood.• We don't have sufficient evidence to say there is a significant difference between meals (p: 0.451).
• However, our best estimate of the meal effect is that changing the meal from BB to SS would increase the number of oocysts in the midgut on average by a factor of e β SS → 1.114.This means that the mean number of oocysts in the midgut is 1.114 times higher when the meal is SS than when the meal is BB with 95% confidence bounds [0.84,1.448].• There is a significant generation effect.
-Generation F1 significantly differs from F5 p: 0.036.This means that the mean number of oocysts in the midgut is 1.442 times higher in generation F1 than in F5, the 95% CI is [1.038,1.983].-Generation F3 significantly differs from F5 p: 0.015.This means that the mean number of oocysts in the midgut is 1.581 times higher in generation F3 than in F5, the 95% CI is

Vector Susceptibility Sensitivity Analysis
Table 7: p-values from the quasi-binomial model comparing, for each generation, the proportion of dissected mosquitoes with oocysts from the two meal groups for Aedes aegypti p-value Generation: F1 0.998 Generation: F3 1.000 Generation: F5 0.654 We found no significant differences in the fraction of infected mosquitoes at any of the three generations.

Anopheles stephensi
• The generation reference is F8.• We performed a likelihood ratio test (LRT) to determine whether there was a significant interaction between generation and meal.The p-value of the test is p: 0.638.• Because of our interaction results we will work with a model that does not include an interaction term.• We don't have sufficient evidence to say there is a significant difference between meals (p: 0.346).
• However, our best estimate of the meal effect is that changing the meal from BB to SS would decrease the number of oocysts in the midgut on average by a factor of e β SS → 0.761.This means that the mean number of oocysts in the midgut is 0.761 times lower when the meal is SS than when the meal is BB with 95% confidence bounds [0.438,1.361].• There is a significant generation effect.Generation F63 significantly differs from F8 (p: <0.001).This means that the mean number of oocysts in the midgut is 9.882 times higher in generation F63 than in F8, the 95% CI is [5.507,17.002].• The plot below presents the observed oocysts counts by generation, along with oocyst count estimates and bootstrap 95% confidence intervals.We found no significant differences in the fraction of infected mosquitoes at any of the three generations.5 Sporozoite Analysis

Analysis Details
• For Aedes aegypti we perform a two-sample t-test on log transformed data and transform the results back to represent the fold-change.For Anopheles stephensi we performed a generalized linear regression with a Gaussian family and fixed effects for meal and generation.• Each considered data point represents a mosquito colony.
• The geometric mean ratio (GMR) is presented for SkitoSnack over bovine blood.

Figure 3 :
Figure3: Anopheles stephensi engorgement rate (%).Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 5 :Figure 6 :
Figure 5: Observed (dots) and estimated (horizontal line) engorgement rates and bootstrap 95% confidence intervals (vertical lines) for Anopheles stephensi.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 7 :
Figure 7: Observed hatch rates (%) for Aedes aegypti.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 9 :
Figure 9: Observed hatch rates for Anopheles stephensi.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 10 :Figure 11 :
Figure 10: Observed (dots) and estimated (horizontal line) hatch rates and bootstrap 95% confidence intervals (vertical lines) for Anopheles stephensi.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.
[1.098,2.315].•The plot below presents the observed oocysts counts by generation, along with oocyst count estimates (dark horizontal line) and bootstrap 95% confidence intervals.

Figure 13 :
Figure 13: Observed (dots) and estimated (horizontal line) oocyst counts and bootstrap 95% confidence intervals (vertical lines) for Aedes aegypti.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 14 :
Figure 14: Observed oocyst counts for Anopheles stephensi.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 15 :
Figure 15: Observed (dots) and estimated (horizontal line) oocyst counts and bootstrap 95% confidence intervals (vertical lines) for Anopheles stephensi.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Figure 16 :
Figure16: Oocyst counts from Plasmodium infections from colonies maintained on bovine blood (BB) or SkitoSnack (SS). A. Oocyst counts in mosquitoes Results from 3-4 biological replicate colonies of Aedes aegypti at generations F1, F3, and F5.B. Results from biological replicate colonies of Anopheles stephensi at generations F8, and F63.Colors represent the data from the different replicate colonies fed on BB (circles), or SS (triangles).Horizontal and vertical lines mark the estimated mean and bootstrap 95% confidence intervals of the oocyst counts.
• An LRT was performed to assess the overall generation effect.

Table 1 :
Aedes aegypti summary table in OR scale

Table 3 :
Anopheles stephensi summary table in OR scale • We found a significant effect due to meal.The odds of a mosquito engorging when the meal is SS are 2.71 (95%CI:[1.4,5.51])times higher than when the meal is SS.

Table 4 :
Aedes aegypti hatch rates model summary Figure8: Hatch rates and 95% bootstrap confidence intervals for Aedes aegypti.Circles and triangles represent the data from the different replicate colonies fed on BB (circles), or SS (triangles), respectively.The colors correspond to the replicates which match the colors used in Additional file 3: Microsoft Excel workbook.

Table 5 :
Anopheles stephensi hatch rate model summary

Table 6 :
Oocysts count estimates and 95% bootstrap confidence intervals for Aedes aegypti

Table 8 :
Oocysts count estimates and 95% bootstrap confidence intervals for Anopheles stephensi

Table 9 :
p-values from the quasi-binomial model comparing, for each generation, the proportion of dissected mosquitoes with oocysts from the two meal groups for Anopheles stephensi

Table 10 :
• The geometric mean in the bovine blood group is 55010.19,and in the SkitoSnack group is 73139.27.• The table below presents results for Aedes aegypti Geometric mean ratio (GMR) and 95% CI of sporozoite counts for Aedes aegypti The sporozoite means by generation and meal are presented in the table below: •

Table 11 :
Sporozoite count geometric means by meal and generation for Anopheles stephensi The table below presents GMR of SS to BB along with 95% CI for Anopheles stephensiTable 12: GMR and 95% CI of sporozoite counts for Anopheles stephensi